Optimised extraction of heterocyclic aromatic amines from blood using hollow fibre membrane liquid-phase microextraction and triple quadrupole mass spectrometry

Heterocyclic aromatic amines (HCA) are carcinogenic mutagens formed during cooking of proteinaceous foods, particularly meat. To assist in the ongoing search for biomarkers of HCA exposure in blood, a method is described for the extraction from human plasma of the most abundant HCAs: 2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) (and its isomer 7,8-DiMeIQx), using Hollow Fibre Membrane Liquid-Phase Microextraction. This technique employs 2.5 cm lengths of porous polypropylene fibres impregnated with organic solvent to facilitate simultaneous extraction from an alkaline aqueous sample into a low volume acidic acceptor phase. This low cost protocol is extensively optimised for fibre length, extraction time, sample pH and volume. Detection is by UPLC-MS/MS using positive mode electrospray ionisation with a 3.4 min runtime, with optimum peak shape, sensitivity and baseline separation being achieved at pH 9.5. To our knowledge this is the first description of HCA chromatography under alkaline conditions. Application of fixed ion ratio tolerances for confirmation of analyte identity is discussed. Assay precision is between 4.5 and 8.8% while lower limits of detection between 2 and 5 pg/mL are below the concentrations postulated for acid-labile HCA-protein adducts in blood.

Uranium halide complexes in ionic liquids: an electrochemical and structural study

The electrochemistry of the salts, [emim](2)[UBr6] and [emim](2)[UO2Br4] ([emim] = 1-ethyl-3-methylimidazolium), has been investigated in both a basic and an acidic bromoaluminate(III) ionic liquid. In the basic ionic liquid, the hexabromo salt undergoes a one-electron reversible reduction process at a stationary glassy carbon disc electrode, while the tetrabromodioxo salt was reduced to a uranium(IV) species by an irreversible two-electron process with the simultaneous transfer of oxide to the ionic liquid. On the other hand, dissolution of either of the salts in an acidic bromoaluminate( III) ionic liquid resulted in the formation of the same electroactive species. The solid state structures of the uranium chloride salts, [emim](2)[UCl6] and [emim](2)[UO2Cl4], have previously been reported, but have now been re-evaluated using a new statistical model developed in our group, to determine the presence or absence of weak hydrogen bonding interactions in the crystalline state.

Brønsted acids in ionic liquids: How acidity depends on the liquid structure

Gutmann Acceptor Number (AN) values have been determined for Brønsted acid–ionic liquid mixtures, over a wide compositional range. Four systems of general formula [C2mim][A]–HA (A− = bistriflamide, [NTf2]−; triflate, [OTf]−; mesylate, [OMs]−; or acetate, [OAc]−, [C2mim]+ = 1-ethyl-3-methylimidazolium cation) were studied. A library of Brønsted acidic systems of varying acidity was constructed and the AN parameter was found to be a convenient approach for quantifying their acidity. HOAc, HOMs and HOTf, when dissolved in ionic liquids, were found to associate with the respective anions to form hydrogen-bonded anionic clusters, [A(HA)x]−. In contrast, HNTf2 was solubilised as a discrete, undissociated molecule. AN values were sensitive to the presence of anionic clusters; acidity could be buffered to a particular AN by binding the solubilised acid in the anionic cluster form. Overall, a simple way to manipulate and quantify the Brønsted acidity of acid–ionic liquid mixtures was demonstrated, and measured AN values were related to liquid speciation.

Macrophage migration inhibitory factor (MIF) expression in human malignant gliomas contributes to immune escape and tumour progression

Macrophage migration inhibitory factor (MIF), which inhibits apoptosis and promotes angiogenesis, is expressed in cancers suppressing immune surveillance. Its biological role in human glioblastoma is, however, only poorly understood. We examined in-vivo expression of MIF in 166 gliomas and 23 normal control brains by immunohistochemistry. MIF immunoreactivity was enhanced in neoplastic astrocytes in WHO grade II glioma and increased significantly in higher tumour grades (III-IV). MIF expression was further assessed in 12 glioma cell lines in vitro. Quantitative RT-PCR showed that MIF mRNA expression was elevated up to 800-fold in malignant glioma cells compared with normal brain. This translated into high protein levels as assessed by immunoblotting of total cell lysates and by ELISA-based measurement of secreted MIF. Wild-type p53-retaining glioma cell lines expressed higher levels of MIF, which may be connected with the previously described role of MIF as a negative regulator of wild-type p53 signalling in tumour cells. Stable knockdown of MIF by shRNA in glioma cells significantly increased tumour cell susceptibility towards NK cell-mediated cytotoxicity. Furthermore, supernatant from mock-transfected cells, but not from MIF knockdown cells, induced downregulation of the activating immune receptor NKG2D on NK and CD8+ T cells. We thus propose that human glioma cell-derived MIF contributes to the immune escape of malignant gliomas by counteracting NK and cytotoxic T-cell-mediated tumour immune surveillance. Considering its further cell-intrinsic and extrinsic tumour-promoting effects and the availability of small molecule inhibitors, MIF seems to be a promising candidate for future glioma therapy.

A new and less destructive laboratory procedure for the physical separation of distal glass tephra shards from sediments

Tephrochronology, a key tool in the correlation of Quaternary sequences, relies on the extraction of tephra shards from sediments for visual identification and high-precision geochemical comparison. A prerequisite for the reliable correlation of tephra layers is that the geochemical composition of glass shards remains unaltered by natural processes (e.g. chemical exchange in the sedimentary environment) and/or by laboratory analytical procedures. However, natural glasses, particularly when in the form of small shards with a high surface to volume ratio, are prone to chemical alteration in both acidic and basic environments. Current techniques for the extraction of distal tephra from sediments involve the ‘cleaning’ of samples in precisely such environments and at elevated temperatures. The acid phase of the ‘cleaning’ process risks alteration of the geochemical signature of the shards, while the basic phase leads to considerable sample loss through dissolution of the silica network. Here, we illustrate the degree of alteration and loss to which distal tephras may be prone, and introduce a less destructive procedure for their extraction. This method is based on stepped heavy liquid flotation and which results in samples of sufficient quality for analysis while preserving their geochemical integrity. In trials, this method out-performed chemical extraction procedures in terms of the number of shards recovered and has resulted in the detection of new tephra layers with low shard concentrations. The implications of this study are highly significant because (i) the current database of distal tephra records and their corresponding geochemical signatures may require refinement and (ii) the record of distal tephras may be incomplete due to sample loss induced by corrosive laboratory procedures. It is therefore vital that less corrosive laboratory procedures are developed to make the detection and classification of distal glass tephra more secure.

Distribution of Uranium and Thorium in Dolomitic Gravel Fill and Shale Saprolite

The objectives of this study were to examine (1) the distribution of U and Th in dolomitic gravel fill and shale saprolite, and (2) the removal of uranium from acidic groundwater by dolomitic gravel through precipitation with amorphous basaluminite at the U.S. DOE Oak Ridge Integrated Field Research Challenge (ORIFRC) field site west of the Oak Ridge Y-12 National Security Complex in East Tennessee. Media reactivity and sustainability are a technical concern with the deployment of any subsurface reactive media. Because the gravel was placed in the subsurface and exposed to contaminated groundwater for over 20 years, it provided a unique opportunity to study the solid and water phase geochemical conditions within the media after this length of exposure. This study illustrates that dolomite gravel can remove U from acidic contaminated groundwater with high levels of Al3+, Ca2+, NO3−, and SO42− over the long term. As the groundwater flows through high pH carbonate gravel, U containing amorphous basaluminite precipitates as the pH increases. This is due to an increase in groundwater pH from 3.2 to ∼6.5 as it comes in contact with the gravel. Therefore, carbonate gravel could be considered as a possible treatment medium for removal and sequestration of U and other pH sensitive metals from acidic contaminated groundwater. Thorium concentrations are also high in the carbonate gravel. Thorium generally shows an inverse relationship with U from the surface down into the deeper saprolite. Barite precipitated in the shallow saprolite directly below the dolomitic gravel from barium present in the acidic contaminated groundwater.

Identification and Functional Analysis of a Novel Tryptophyllin Peptide from the Skin of the Red-eye Leaf Frog, Agalychnis callidryas

Amphibian skin has proved repeatedly to be a largely untapped source of bioactive peptides and this is especially true of members of the Phyllomedusinae subfamily of frogs native to South and Central America. Tryptophyllins are a group of peptides mainly found in the skin of members of this genus. In this study, a novel tryptophyllin (TPH) type 3 peptide, named AcT-3, has been isolated and structurally-characterised from the skin secretion and lyophilised skin extract of the red-eye leaf frog, Agalychnis callidryas. The peptide was identified in and purified from the skin secretion by reverse-phase HPLC. MALDI-TOF mass spectrometry and MS/MS fragmentation sequencing established its primary structure as: pGlu-Gly-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-Phe-Leu-Pro-Glu, with a non-protonated molecular mass of 1538.19Da. The mature peptide possessed the canonical N-terminal pGlu residue that arises from post-translational modification of a Gln residue. The deduced open-reading frame consisted of 63 amino acid residues encoding a highly-conserved signal peptide of approximately 22 amino acid residues, an intervening acidic spacer peptide domain, a single AcT-3 encoding domain and a C terminal processing site. A synthetic replicate of AcT-3 was found to antagonise the effect of BK on rat tail artery smooth muscle and to contract the intestinal smooth muscle preparations. It was also found that AcT-3 could dose-dependently inhibit the proliferation of human prostate cancer cell lines after 72h incubation.

Air pollutant contamination and acidification of surface waters in the North York Moors, UK: Multi-proxy evidence from the sediments of a moorland pool

Despite the extensive geographical range of palaeolimnological studies designed to assess the extent of surface water acidification in the United Kingdom during the 1980s, little attention was paid to the status of surface waters in the North York Moors (NYM). In this paper, we present sediment core data from a moorland pool in the NYM that provide a record of air pollution contamination and surface water acidification. The 41-cm-long core was divided into three lithostratigraphic units. The lower two comprise peaty soils and peats, respectively, that date to between approximately 8080 and 6740 cal. BP. The uppermost unit comprises peaty lake muds dating from between approximately ad 1790 and the present day (ad 2006). The lower two units contain pollen dominated by forest taxa, whereas the uppermost unit contains pollen indicative of open landscape conditions similar to those of the present. Heavy metal, spheroidal carbonaceous particle, mineral magnetics and stable isotope analysis of the upper sediments show clear evidence of contamination by air pollutants derived from fossil-fuel combustion over the last c. 150years, and diatom analysis indicates that the naturally acidic pool became more acidic during the 20th century. We conclude that the exceptionally acidic surface waters of the pool at present (pH=c. 4.1) are the result of a long history of air pollution and not because of naturally acidic local conditions. We argue that the highly acidic surface waters elsewhere in the NYM are similarly acidified and that the lack of evidence of significant recovery from acidification, despite major reductions in the emissions of acidic gases that have taken place over the last c. 30years, indicates the continuing influence of pollutant sulphur stored in catchment peats, a legacy of over 150years of acid deposition.

The effect of pH on the photonic efficiency of the destruction of methyl orange under controlled periodic illumination with UV-LED sources

The nature of photon interaction and reaction pH can have significant impacts on semiconductor photocatalysis. This paper describes the effect of pH on the photonic efficiency of photocatalytic reactions in the aqueous phase using TiO2 catalysts. The reactor was irradiated using periodic illumination with UV-LEDs through control of the illumination duty cycle (γ) through a series of light and dark times (Ton/Toff). Photonic efficiencies for methyl orange degradation were found to be comparable at high γ irrespective of pH. At lower γ, pH effects on photonic efficiency were very distinct across acidic, neutral and alkaline pH indicating an effect of complementary parameters. The results suggest photonic efficiency is greatest as illumination time, Ton approaches interfacial electron-transfer characteristic time which is within the range of this study or charge-carrier lifetimes upon extrapolation and also when electrostatic attraction between surface-trapped holes, {TiIVOH}ads+ and substrate molecules is strongest.

Synthesis of visible-light-activated yellow amorphous TiO2 photocatalyst

Visible-light-activated yellow amorphous TiO₂ (yam- TiO ₂) was synthesised by a simple and organic-free precipitation method. TiN, an alternative precursor for TiO₂ preparation, was dissolved in hydrogen peroxide under acidic condition (pH∼1) adjusted by nitric acid. The yellow precipitate was obtained after adjusting pH of the resultant red brown solution to 2 with NH₄OH. The BET surface area of this sample was 261 m²/g. The visible light photoactivity was evaluated on the basis of the photobleaching of methylene blue (MB) in an aqueous solution by using a 250 W metal halide bulb equipped with UV cutoff filter (λ>420 nm) under aerobic conditions. Yam- TiO₂ exhibits an interesting property of being both surface adsorbent and photoactive under visible light. It was assigned to the η²-peroxide, an active intermediate form of the addition of H₂O₂ into crystallined TiO₂ photocatalyst. It can be concluded that an active intermediate form of titanium peroxo species in photocatalytic process can be synthesised and used as a visible-light-driven photocatalyst

Convergence of products from Povarov and von Miller reactions: approaches to helquinoline

Yttrium triflate or triflic acid catalysed Povarov reaction of methyl anthranilate with ethyl vinyl ether, both as aldehyde surrogate and as alkene, gave the desired 2-methyl-4-ethoxytetrahydroquinoline diastereoisomers as the major products along with four component coupling von Miller adducts. A proton NMR-study, using yttrium triflate as catalyst, revealed that the cis-diastereoisomers were the initial major products in both the Povarov and von Miller reactions but that these isomerised to the trans-diastereoisomers under the reaction conditions. Two distinct pathways for forming von Miller adducts were uncovered with the initial Povarov products being converted to von Miller adducts under the reaction conditions. Replacement of the 4-ethoxy with a 4-methoxy group under acidic conditions gave predominantly the trans-diastereoisomer, which was subsequently converted to a cis/trans mixture of the tetrahydroquinoline antibiotic helquinoline. It was also possible to convert the von Miller products to Povarov products under acidic conditions

Development of a PtSn bimetallic catalyst for direct fuel cells using bio-butanol fuel

Pt and PtSn catalysts were studied for n-butanol electro-oxidation at various temperatures. PtSn showed a higher activity towards butanol electro-oxidation compared to Pt in acidic media. The onset potential for n-butanol oxidation on PtSn is ~520 mV lower than that found on Pt, and significantly lower activation energy was found for PtSn compared with that for Pt.

Evidence supporting a role for N-epsilon-(3-formyl-3,4-dehydropiperidino)lysine accumulation in Muller glia dysfunction and death in diabetic retinopathy

Purpose: Recent evidence suggests that neuroglial dysfunction and degeneration contributes to the etiology and progression of diabetic retinopathy. Advanced lipoxidation end products (ALEs) have been implicated in the pathology of various diseases, including diabetes and several neurodegenerative disorders. The purpose of the present study was to investigate the possible link between the accumulation of ALEs and neuroretinal changes in diabetic retinopathy.

Methods: Retinal sections obtained from diabetic rats and age-matched controls were processed for immunohistochemistry using antibodies against several well defined ALEs. In vitro experiments were also performed using a human Muller (Moorfields/Institute of Ophthalmology-Muller 1 [ MIO-M1]) glia cell line. Western blot analysis was used to measure the accumulation of the acrolein-derived ALE adduct N epsilon-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) in Muller cells preincubated with FDP-lysine-modified human serum albumin (FDP-lysine-HSA). Responses of Muller cells to FDP-lysine accumulation were investigated by analyzing changes in the protein expression of heme oxygenase-1 (HO-1), glial fibrillary acidic protein (GFAP), and the inwardly rectifying potassium channel Kir4.1. In addition, mRNA expression levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF alpha) were determined by reverse transcriptase PCR (RT-PCR). Apoptotic cell death was evaluated by fluorescence-activated cell sorting (FACS) analysis after staining with fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide.

Results: No significant differences in the levels of malondialdehyde-, 4-hydroxy-2-nonenal-, and 4-hydroxyhexenal-derived ALEs were evident between control and diabetic retinas after 4 months of diabetes. By contrast, FDP-lysine immunoreactivity was markedly increased in the Muller glia of diabetic rats. Time-course studies revealed that FDP-lysine initially accumulated within Muller glial end feet after only a few months of diabetes and thereafter spread distally throughout their inner radial processes. Exposure of human Muller glia to FDP-lysine-HSA led to a concentration-dependent accumulation of FDP-lysine-modified proteins across a broad molecular mass range. FDP-lysine accumulation was associated with the induction of HO-1, no change in GFAP, a decrease in protein levels of the potassium channel subunit Kir4.1, and upregulation of transcripts for VEGF, IL-6, and TNF-alpha. Incubation of Muller glia with FDP-lysine-HSA also caused apoptosis at high concentrations.

Conclusions: Collectively, these data strongly suggest that FDP-lysine accumulation could be a major factor contributing to the Muller glial abnormalities occurring in the early stages of diabetic retinopathy.

Design of an automated solar concentrator for the pyrolysis of scrap rubber

An automated solar reactor system was designed and built to carry out catalytic pyrolysis of scrap rubber tires at 550°C. To maximize solar energy concentration, a two degrees-of-freedom automated sun tracking system was developed and implemented. Both the azimuth and zenith angles were controlled via feedback from six photo-resistors positioned on a Fresnel lens. The pyrolysis of rubber tires was tested with the presence of two types of acidic catalysts, H-beta and H-USY. Additionally, a photoactive TiO<inf>2</inf> catalyst was used and the products were compared in terms of gas yields and composition. The catalysts were characterized by BET analysis and the pyrolysis gases and liquids were analyzed using GC-MS. The oil and gas yields were relatively high with the highest gas yield reaching 32.8% with H-beta catalyst while TiO<inf>2</inf> gave the same results as thermal pyrolysis without any catalyst. In the presence of zeolites, the dominant gasoline-like components in the gas were propene and cyclobutene. The TiO<inf>2</inf> and non-catalytic experiments produced a gas containing gasoline-like products of mainly isoprene (76.4% and 88.4% respectively). As for the liquids they were composed of numerous components spread over a wide distribution of C<inf>10</inf> to C<inf>29</inf> hydrocarbons of naphthalene and cyclohexane/ene derivatives.

Contamination of Donana food-chains after the Aznalcollar mine disaster

On 25 April 1998 part of the tailings pond dike of the Aznalcollar Zn mine north of the Guadalquivir marshes (Donana) in southern Spain collapsed releasing an estimated 5 million m³ of acidic metal-rich waste. This event contaminated farmland and wetland up to >40 km downstream, including the 900-ha 'Entremuros', an important area for birds within the Donana world heritage site. In spite of the contamination, birds continued to feed in this area. Samples of two abundant macrophytes (Typha dominguensis and Scirpus maritimus) were taken from the Entremuros and nearby uncontaminated areas; these plants are important food items for several bird species. Analyses showed that in the Entremuros mean plant tissue concentrations of Cd were 3-40-fold (0.8-7.4 ppm) and Zn 20-100-fold (20-3384 ppm) greater than those from control areas. Comparable dietary concentrations of Zn have been reported to cause severe physiological damage to aquatic birds under experimental conditions. Elevated Cd concentrations are of concern as Cd bioconcentrates and is a cumulative poison. Metals released in this accident are moving into this food-chain and present a considerable risk to species feeding on Typha sp. and Scirpus sp. Many other food-webs exist in this area and require detailed examination to identify the species at risk, and to facilitate the management of these risks to minimise future impacts to the wildlife of Donana. Copyright (C) 1999 Elsevier Science Ltd.